The present invention relates to denaturant and protease stable proteins, modified derivatives thereof, and uses thereof. More particularly, the present invention relates to the use of novel denaturant-stable, protease resistant, homo-oligomeric proteins, also referred to herein as stable proteins (SPs), and derivatives thereof designed for complexing, release and delivery of other molecules (ligands) and nanostructures.
Denaturant-Stable, Protease Resistant Proteins
A unique family of stress-induced, chaperone-like proteins having exceptional resistance to harsh conditions has been recently identified in widely diverse plant species. Exemplified by the SP1 protein of Aspen (SEQ ID NO: 1), this family of proteins is characterized by boiling-, denaturant- and protease-resistance, regions of conserved amino acid sequence homology, unique three-dimensional conformation, oligomer formation and a strong stabilizing effect on biologically active proteins.
The exceptional resistance of these stress-induced, chaperone-like proteins to harsh conditions in combination with their unique three dimensional structure allows the application of extreme condition to create stable, but selectively reversible complexes with the ligand.
SP1
SP1, isolated from aspen plants (Populus tremula), responds to a wide range of environmental stresses, including salinity, cold and heat stress and accumulates during stress recovery. No significant sequence similarity has been found with known protein families and SP1 homologues have been observed in a number of plant and bacterial species, either as putative proteins from genomic sequences or ESTs with unknown function.
Wang et al. (U.S. patent application Ser. No. 10/233,409) have isolated, cloned and characterized the Aspen SP1 protein (SEQ ID NO: 1), and uncovered it's chaperone-like activity in stabilizing other, biologically active proteins against denaturation. Wang et al (U.S. patent application Ser. No. 10/233,409) further disclosed other boiling and detergent-stable proteins from other, diverse plant species (Tomato, Pine, Rice, Corn and Arabidopsis) sharing similar functional characteristics, specifically, chaperone-like activity and stress-relatedness, sharing immune-cross reactivity, having at least 65% amino acid homology to the Aspen SP1, and sharing a conserved region of sequence homology.
Wang et al (U.S. patent application Ser. No. 10/233,409) disclosed SP1 proteins fused to other protein or non-protein molecules, for enhancement of binding properties of binding molecules, for stabilization of the fused molecules (such as enzymes) and for enhancement or alteration of immunological properties of the fused molecules. SP1 fusion proteins, as taught by 10/233,409, comprise recombinant SP1 molecules having additional polypeptide sequences added by genetic engineering techniques, and SP1 molecules having additional non-protein moieties added by chemical means, such as cross linking. Wang et al have further disclosed the therapeutic use of SP1 proteins for strengthening skin, hair, nails, etc. However, U.S. patent application Ser. No. 10/233,409 do not teach, nor imply, the use of native SP1, or SP1 variants as carriers for and means of controlled release of, agents (therapeutic, cosmetic, diagnostic, conductive, etc) reversibly complexed therewith.
Drug Carriers:
Many drugs employed to treat diseases are either insufficiently soluble in aqueous solutions or have adverse side effects in therapeutic concentrations. Thus, many medical applications suffer from a lack of suitable methods for efficiently delivery of effective concentrations of drugs to a target cell or tissue in an organism (e.g., mammal) in need of treatment.
Some considerations for efficacious use of drugs include:
Poor solubility, causing difficulty in achieving a convenient pharmaceutical format, as hydrophobic drugs may precipitate in aqueous media. However, the use of excipients for solubilization such as Cremphor (the solubilizer for paclitaxel in Taxol) is also associated with toxicity.
Lack of selectivity for target tissues, leading to toxicity to normal tissues, severely restricting the amount of drug that can be administered, as in the case of the cardiac toxicity of doxorubicin. Low concentrations of drugs in target tissues further results in suboptimal therapeutic effects.
Unfavorable pharmacokinetics, such as rapid renal clearance, rapid breakdown of the drug in vivo, or loss of activity at physiological conditions (e.g. loss of activity of camptothecins at physiological pH), can also lead to heightened dosing or a frequent administration regimen.
Development of drug resistance in target tissue, such as tumors, by induction of cellular transporters, detoxification pathways, or inhibition of apoptosis transduction pathways.
Tissue damage on extravasation of cytotoxic drugs, leading to tissue damage (i.e. necrosis caused by free paclitaxel).
A number of approaches have resolved some of these issues in specific cases, but there is yet no general solution to the problems of drug delivery. Some examples of existing approaches for solving these problems include (1) solublization of hydrophobic drugs in micelles formed from surfactants in aqueous media (Wiedmann and Kamel, J. Pharm. Sci. 2002, 91, 1743; MacGregor, et al., Adv. Drug Deliv. Rev. 1997, 25, 33), (2) encapsulation of drugs in polymeric matrices in the nanometer to micrometer size range which may be biodegradable and may contain bioadhesive functional groups or ligands (WO 02/15877, WO 02/49676), (3) encapsulation of hydrophilic drugs in liposomes (Anderson, et al., Pharm. Res. 2001, 18, 316; WO 99/33940), which may also display bioadhesive functional groups or ligands, (4) conjugation of drugs to molecules that are substrates for active transport systems (Kramer, et al., J. Biol. Chem. 1994, 269, 10621; WO 01/09163; US 2002/0098999; US 20060074225), (5) targeting using physiologically selective (pH, enzymatic, etc.) release of active drug components (i.e. prodrugs), (6) association of the drug with hydrogels and (7) chemical derivatization of protein drugs with hydrophilic polymers to protect them from degradation, immune recognition, or renal excretion (Belcheva, et al., Bioconjugate Chem. 1999, 10, 932; Zalipsky, Bioconjugate Chem. 1995, 6, 150; U.S. Pat. No. 4,002,531; U.S. Pat. No. 4,179,337). None of these approaches, however, offers a general solution for all cases of drug delivery problems. Control of particle size in micellar, liposomal, and polymeric nanoparticulate systems remains a serious problem. The inability of currently available drug delivery systems to incorporate all of the functions required for delivery into a single system is another problem with for example, micelles, nanoparticulate systems and targeted systems. Yet further, the release rate and storage life, especially of micelles and liposomes, is difficult to control and unpredictable, and amphiphylic components can produce toxic effects.
Other systems employed for drug delivery to a cell or tissue of an organism have similar drawbacks. Thus, there is a need for a method to deliver drugs that minimize or overcome the above-referenced problems.
The invention includes methods for the use of SP1 and SP1 variants for forming molecular complexes with other substances such as small molecules, peptides, nucleic acid fragments, inorganic nanostructures and other molecules (ligands). In addition the invention includes methods for the use of SP1 and SP1 variants for molecular complexing of drugs and delivery as well as control release of complexed ligands. There is thus a widely recognized need for, and it would be highly advantageous to have, SP1 and SP1 variants capable of forming molecular complexes devoid of the above limitation.